Summary: 1.Orthopoxvirus pathogenesis A goal of EVPS is to understand virus-host interactions and exploit them for countermeasure development. Previously we had focused on nonhuman primate model development, these experiments are completed and have been published or are pending. Our continuing orthopoxvirus work involves Backwards Matched Longitudinal Analysis which identified several cytokines that were statistically associated with lethal or non-lethal outcome in NHPs. The duration and intensity of certain cytokines was associated with survival (IFN-gamma and RANTES) or non-survival (MCP-1). The role of these cytokines was investigated using recombinant virus expressing each cytokine and knockout mice of the cytokine or its receptor. MCP-1 expressing virus and MCP-1 and MCP-1 receptor knockout mice had increased pathogenicity when compared to wild type virus. The MCP-1data suggests that macrophages are essential for controlling infection, but excessive MCP-1 alter macrophage function which may exacerbate disease. IFN-gamma expressing virus did not develop disease, and knockout of IFN-gamma or its receptor resulted in increased pathogenicity. Mice inoculated with interferon gamma expressing virus did not develop any signs of disease and survived lethal challenge at the equivalent 100% lethal dose of wild type virus. Expression of interferon gamma at the site of infection via the recombinant virus resulted in protection from challenge with the LD100 of wild type virus. 2. Bivalent vaccines that confer protection against rabies and Ebola virus We have been developing a rabies virus based vaccine that expresses the glycoprotein (GP) from ebolavirus, marburgvirus and sudanvirus. Previously we developed (a) replication-competent, (b) replication-deficient, and (c) chemically inactivated rabies virus (RABV) vaccines expressing Zaire ebolavirus (EBOV) glycoprotein (GP) using a reverse genetics system based on the SAD B19 RABV wildlife vaccine in collaboration with Matthias Schnell of Thomas Jefferson University. Immunization with live or inactivated RABV vaccines expressing ZEBOV GP induced cellular and humoral immunity against each virus and conferred protection from both lethal RABV and EBOV challenge in mice. We evaluated our vaccine candidates in a rhesus macaque challenge model. 100% protection was observed with live attenuated RABV-GP. The inactivated and RVdelG-GP viruses provided 50% protection. Strong humoral and cellular immunity was observed. In summary, our findings indicated that RV-GP retains the attenuation phenotype of the live-attenuated RABV vaccine, and RVdelG-GP would appear to be an even safer alternative for use in wildlife or consideration for human use. We also further developed our vaccine constructs to use codon optimized filoviruses and have demonstrated immunogenicity and safety of these constructs in mice. We have evaluated the codon optimized rabies-ebolavirus construct and demonstrated 83% protection in the absence of an adjuvant and 100% protection in the presence of an adjuvant. Studies are ongoing to shift from a 2 dose schedule to a 3 dose schedule to reflect the human rabies virus vaccine schedule. A clinical trial lot of the rabies-ebolavirus has been produced and a Phase I clinical trial is expected to start in the Fall of 2015. The rabies-Marburg construct has demonstrated immunogenicity in mice and efficacy testing is scheduled to start in the Fall of 2015 in the mouse and nonhuman primate models of Marburg virus. 3.Filovirus Molecular Virology We are also investigating molecular virology of filoviruses. We are studying the role of phosphorylation of the viral proteins in the virus lifecycle. We have chosen to focus on VP35 as it is essential for transcription and replication of the genome. Using a replicon system we have generated point mutations and clustered point mutations of potential phosphorylation sites and evaluate the mutant for replication and transcription. Our data indicates a hierarchical phosphorylation strategy that regulates VP35 function. A second project involves identification of structure and function relationships of the EBOV noncoding regions. We have identified host proteins that interact with these EBOV genome regions. We are working to identify the proteins. We have identified HSC70 as interacting with at least one domain in the EBOV Trailer by pull-down assays and confirmed using immunoprecipitation-RT-PCR. Three HSC70 binding domains have been identified within the EBOV Trailer and mutational analysis of the 5 most region results in a decrease in replicon signal. We have complete SHAPE analysis of the EBOV Trailer and Leader to define the secondary structure of these untranslated regions. Based on these data we may identify therapeutic targets as well as establish mechanisms of filovirus lifecycle regulation. 4. MERS-CoV We have also been developing NHP models for MERS-CoV. We have established that intratracheal exposure of rhesus monkeys with MERS-CoV Jordan isolate results in limited respiratory tract disease that peaks between days 4 and 5 post inoculation, resolves by day 9 post inoculation and can be quantified by computed tomography. Inoculation of common marmosets resulted in a longer lived respiratory tract disease that peaks between days 3 and 14, but does not completely resolve, and can be monitored and quantified by computed tomography. Furthermore, marmosets exposed to media alone or gamma-irradiated, inactivated virus develop similar disease as marmosets exposed to infectious virus. Infectious virus could not be recovered from serial samples in either rhesus or common marmosets. To further develop the MERS-CoV model we are working with Dr. Ralph Baric to construct and evaluate a MERS-CoV that expresses the human sodium-iodide symporter that can be used as a PET reporter. We expect this recombinant virus to provide enhanced detail on the infectious process of MERS in rabbits and NHPs. We have also demonstrated efficacy of a transchromatic cow generated, fully humanized polyclonal antibody against MERS-CoV in the mouse hAd-DPP4 model of MERS developed in collaboration with SAB and NMRC.